This work was fully supported by the NHLBI intramural program (Project # ZIA HL00460131).

All animal protocols were approved by the National Heart, Lung, and Blood Institute Animal Care and Use Committee and performed in accordance with the guidelines described in the Animal Care and Welfare Act (7 USC 2142 &#167; 13).
1. Isolated Perfused Heart System and Perfusate
NOTE: This preparation is very similar to previous publications23.
<ol>
	<li>Make 4 liters of modified Krebs-Henseleit perfusate composed of (in mmol/L) 137.0 NaCl, 5.4 KCl, 1.8 CaCl2, 0.5 MgCl2, 1.0 Na2HPO4, 10.0 glucose, 1.0 lactate, and 10.0 HEPES.</li>
	<li>pH the perfusate to 7.4 at 37 &#176;C with NaOH and HCl.</li>
	<li>Filter the perfusate through a 1 &#181;m pore membrane.</li>
	<li>Rinse all tubes and chambers of the perfused heart system by running and draining purified water through the system.</li>
	<li>Transfer the perfusate into the tank and oxygenate with 100% O2 with the bubbler while maintaining the temperature at 37 &#176;C using a heated circulating water bath.</li>
	<li>Add 2 of 12 &#181;m pore membrane filters and prime the system with the perfusate while recirculating in the Langendorff mode.</li>
	<li>Attach a tubing clamp on the tube right above the aortic cannula and adjust the screw so that the aortic flow drops to about 10 mL/min.</li>
</ol>
2. Rabbit Heart Excision and Perfusion
<ol>
	<li>Heart excision
	<ol>
		<li>Anesthetize male New Zealand white rabbits (about 3 kg) via a 1.5 mL intramuscular injection of ketamine/acepromazine mixture (10:1).</li>
		<li>Approximately 10&#8211;15 minutes later, administer 3% isoflurane via inhalation for a complete anesthetic effect.</li>
		<li>Confirm proper depth of anesthesia by toe pinching and then place a line in the marginal ear vein for administration of subsequent drugs.</li>
		<li>Inject 1,500 units (or 1.5 mL of 1,000 units/mL) of Heparin and let circulate for 3 minutes.</li>
		<li>Double check proper depth of anesthesia and then euthanize with 6 mEq (or 3 mL of 2 mEq/mL) of KCl.</li>
		<li>Rapidly open the chest, locate the heart&#8217;s apex and the aorta. Remove the heart by cutting the aorta as far from the heart as possible and cutting the pulmonary veins as close to the lungs as possible. 
		&#8203;NOTE: Removal of lungs at this early stage is different than previous publication23 but has not impact on preparation.</li>
		<li>Place the heart in a small beaker of perfusate (same perfusate as step 1.3) sitting in a bucket of ice for transportation from surgery to perfusion.</li>
	</ol>
	</li>
	<li>Heart cannulation
	<ol>
		<li>Cannulate and tie the aorta securely, making sure not to include any bubbles in the aortic line.</li>
		<li>Initiate flow at 70 mmHg perfusion pressure by removing the tubing clamp on the aortic line and maintain this pressure during the remainder of the surgery and vessel cannulation.</li>
		<li>Separate the pulmonary artery from the aorta and other vessels and ligate the vena cavae and pulmonary veins. Remove the fat and connective tissue still present.</li>
		<li>Cannulate the pulmonary artery to provide a measure of coronary sinus flow rate and oxygen tension.</li>
		<li>Discard initial flow out of the heart (for about 10 minutes) during the preparation to eliminate blood and surgical debris. After this period, recirculate the perfusate.</li>
	</ol>
	</li>
</ol>
3. Side-Firing Fiber Optic Placement
<ol>
	<li>Connect the fiber optic catheter to a high-power fiber-coupled LED white light source to help visualization as well as provide the light for spectroscopy once in the heart.</li>
	<li>Cut a small appendage of the left atrium, insert the catheter into the left ventricle via the mitral valve, then rotate it to achieve an illuminated left ventricle free wall.</li>
	<li>Position the pickup fiber optic directly opposite the region of maximum illumination of the left ventricle at about 1 cm from the heart.</li>
	<li>Connect the other end of the pickup fiber to a rapid scanning spectrometer.</li>
</ol>
4. Optical Spectroscopy
<ol>
	<li>Turn off the lights in the experimental area to obtain complete darkness.</li>
	<li>Start the custom program, incorporating spectrometer drivers to perform data acquisition and real-time analysis of the transmitted light. 
	&#8203;NOTE: An executable version of the consolidated version of the spectral acquisition and analysis program is provided as a Supplemental Coding File. Source code is available by request to the authors.</li>
	<li>Navigate through all prompts, selecting options for perfused heart spectroscopy acquisition mode. On the next page, indicate whether auxiliary data collection is occurring. Finally, enter acquisition parameters, including location of both chromophores reference spectra and data to be saved.</li>
	<li>Enter a bandwidth of 490&#8211;630 nm.</li>
	<li>Enter a sampling rate of 2 Hz (i.e. 2 samples/sec).</li>
	<li>Collect a dark current, or zero light, spectrum to correct for background signal levels by turning the light source off.</li>
	<li>Click to select the chromophore references desired to be used in the fitting routine.</li>
	<li>In the Acquire Data page, adjust the position of both the catheter and the pickup fiber to maximize the transmitted light displayed on the software with specific attention to the signal amplitude in the 500 nm region, where the oxygenated myoglobin absorbances should be observed.</li>
	<li>Make sure the transmitted light is not saturating the detector in the 600 nm region.</li>
	<li>Ensure no external light sources contribute to the collected spectrum by turning off catheter illumination and confirming no light is now detected.</li>
	<li>Initiate the data collection by clicking on the Save Spectra button.</li>
	<li>Click on Set as Control to view the difference absorbance spectrum from future spectra to the current &#8220;control&#8221; spectrum.</li>
	<li>Perform any physiological perturbation as desired.
	<ol>
		<li>Protocol 1: Effect of Cyanide on Cardiac Performance and Chromophore Absorption
		<ol>
			<li>Stop recirculating fluid from the heart perfusion.</li>
			<li>Using a syringe pump, inject cyanide (2.5 to 75 mM at pH 7) at different rates into perfusate just before the aortic cannula to achieve desired concentrations of cyanide (0.025 to 1 mM, calculated from aortic flow rate) in the flowing perfusate into the heart while monitoring cardiac function and optical properties.</li>
			<li>Stop the cyanide syringe pump when the effects on coronary flow and heart rate along with the optical transmission through the heart wall are at steady state.</li>
		</ol>
		</li>
		<li>Protocol 2: Ischemia/Hypoxia
		<ol>
			<li>Stop cyanide infusion.</li>
			<li>After 5 minutes switch the bubbling gas from 100% oxygen to 100% nitrogen to remove oxygen from the system.</li>
			<li>After about 10 minutes, stop the flow to simulate a total ischemic/hypoxic condition.</li>
		</ol>
		</li>
	</ol>
	</li>
</ol>
5. Spectral data analysis
<ol>
	<li>Run the program in the perfused heart analysis mode.</li>
	<li>Select appropriate spectrometer.</li>
	<li>Enter the data file path and reference spectra file and select catheter light source, which loads the pre-saved spectrum of the catheter light source.</li>
	<li>Select Read Bin Data. &#160;</li>
	<li>Select Set Min and Max Wavelength.</li>
	<li>Enter the bandwidth for the data analysis as 490&#8211;630 nm.</li>
	<li>Select Return to Main Menu.</li>
	<li>Select Read References.</li>
	<li>Confirm the reference spectra to use in the analysis.</li>
	<li>Select Return to Main Menu.</li>
	<li>Select Time Points in the main menu.</li>
	<li>Select a T0 time point as control and set the range to 100 points.</li>
	<li>Select a T1 time point as the experimental period at a range of 100 points.</li>
	<li>Observe the raw difference spectrum in the Averaged Abs. Spectrum tab.</li>
	<li>Select Calculate Fit Coefficients and then click on the Fit Coefficients tab to observe the time course of the reference spectra fit.</li>
	<li>Return to the Main Menu and select Calculate Difference Abs.</li>
	<li>Select T0 and &#916;T1 at all positions. Observe the fitted spectrum in the Difference Spectrum window and fitting elements in the Reference Weight window.</li>
	<li>Repeat this procedure to compare other time points in the experiment.</li>
	<li>Return to the Main Menu.</li>
	<li>Save data and analysis in a spreadsheet report by typing in a desired name and selecting Save Data for further analysis with other programs. 
	NOTE: if no name is typed, the report is saved with the same name as the input file. The report is saved in a folder named Excel Analysis Files, located in the same folder as the original input file.</li>
</ol>

<ol>
	<li>Arai, A. E., Kasserra, C. E., Territo, P. R., Gandjbakhche, A. H., Balaban, R. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Myocardial+oxygenation+in+vivo:+optical+spectroscopy+of+cytoplasmic+myoglobin+and+mitochondrial+cytochromes.">Myocardial oxygenation in vivo: optical spectroscopy of cytoplasmic myoglobin and mitochondrial cytochromes.</a> American Journal of Physiology. 277, 683-697 (1999).</li><li>Epstein, F. H., Balaban, R. S., Ross, B. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Redox+state+of+cytochrome+aa3+in+isolated+perfused+rat+kidney.">Redox state of cytochrome aa3 in isolated perfused rat kidney.</a> American Journal of Physiology. 243 (4), 356-363 (1982).</li><li>Hassinen, I. E., Hiltunen, J. K., Takala, T. E. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reflectance+spectrophotometric+monitoring+of+the+isolated+perfused+heart+as+a+method+of+measuring+the+oxidation-reduction+state+of+cytochromes+and+oxygenation+of+myoglobin.">Reflectance spectrophotometric monitoring of the isolated perfused heart as a method of measuring the oxidation-reduction state of cytochromes and oxygenation of myoglobin.</a> Cardiovascular Research. 15, 86-91 (1981).</li><li>Makino, N., Kanaide, H., Yoshimura, R., Nakamura, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Myoglobin+oxygenation+remains+constant+during+the+cardiac+cycle.">Myoglobin oxygenation remains constant during the cardiac cycle.</a> American Journal of Physiology. 245 (14), 237-243 (1983).</li><li>Takahashi, E., Doi, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Visualization+of+oxygen+level+inside+a+single+cardiac+myocyte.">Visualization of oxygen level inside a single cardiac myocyte.</a> American Journal of Physiology. 268, 2561-2568 (1995).</li><li>Heineman, F. W., Kupriyanov, V. V., Marshall, R., Fralix, T. A., Balaban, R. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Myocardial+oxygenation+in+the+isolated+working+rabbit+heart+as+a+function+of+work.">Myocardial oxygenation in the isolated working rabbit heart as a function of work.</a> American Journal of Physiology. 262, 255-267 (1992).</li><li>Arakaki, L. S., Burns, D. H., Kushmerick, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Accurate+myoglobin+oxygen+saturation+by+optical+spectroscopy+measured+in+blood-perfused+rat+muscle.">Accurate myoglobin oxygen saturation by optical spectroscopy measured in blood-perfused rat muscle.</a> Applied Spectroscopy. 61 (9), 978-985 (2007).</li><li>Bose, S., French, S., Evans, F. J., Joubert, F., Balaban, R. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Metabolic+network+control+of+oxidative+phosphorylation:+multiple+roles+of+inorganic+phosphate.">Metabolic network control of oxidative phosphorylation: multiple roles of inorganic phosphate.</a> Journal of Biological Chemistry. 278 (40), 39155-39165 (2003).</li><li>Tamura, M., Oshino, N., Chance, B., Silver, I. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optical+measurements+of+intracellular+oxygen+concentrations+of+rat+heart+in+vitro.">Optical measurements of intracellular oxygen concentrations of rat heart in vitro.</a> Archives of Biochemistry and Biophysics. 191, 18-22 (1978).</li><li>Wright, T. J., Davis, R. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Myoglobin+oxygen+affinity+in+aquatic+and+terrestrial+birds+and+mammals.">Myoglobin oxygen affinity in aquatic and terrestrial birds and mammals.</a> The Journal of Experimental Biology. 218, 2180-2189 (2015).</li><li>Wright, T. J., Davis, R. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Myoglobin+extraction+from+mammalian+skeletal+muscle+and+oxygen+affinity+determination+under+physiological+conditions.">Myoglobin extraction from mammalian skeletal muscle and oxygen affinity determination under physiological conditions.</a> Protein Expression and Purification. 107, 50-55 (2015).</li><li>Shibata, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Relationship+between+oxygen+affinity+and+autoxidation+of+myoglobin.">Relationship between oxygen affinity and autoxidation of myoglobin.</a> Inorganic Chemistry. 51 (21), 11955-11960 (2012).</li><li>Kim, N., Ripple, M. O., Springett, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Measurement+of+the+mitochondrial+membrane+potential+and+pH+gradient+from+the+redox+poise+of+the+hemes+of+the+bc1+complex.">Measurement of the mitochondrial membrane potential and pH gradient from the redox poise of the hemes of the bc1 complex.</a> Biophysical Journal. 102 (5), 1194-1203 (2012).</li><li>Oshino, N., Jamieson, D., Sugano, T., Chance, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mitochondrial+function+under+hypoxic+conditions:+The+steady+states+of+cytochrome+a,a3+and+their+relation+to+mitochondrial+energy+states.">Mitochondrial function under hypoxic conditions: The steady states of cytochrome a,a3 and their relation to mitochondrial energy states.</a> Biochimica et Biophysica Acta. 368, 298-310 (1974).</li><li>Glancy, B., Willis, W. T., Chess, D. J., Balaban, R. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effect+of+calcium+on+the+oxidative+phosphorylation+cascade+in+skeletal+muscle+mitochondria.">Effect of calcium on the oxidative phosphorylation cascade in skeletal muscle mitochondria.</a> Biochemistry. 52 (16), 2793-2809 (2013).</li><li>Figulla, H. R., Hoffmann, J., Lubbers, D. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evaluation+of+reflection+spectra+of+the+isolated+heart+by+multicomponent+spectra+analysis+in+comparison+to+other+evaluating+methods.">Evaluation of reflection spectra of the isolated heart by multicomponent spectra analysis in comparison to other evaluating methods.</a> Advances in Experimental Medicine and Biology. 169, 821-830 (1984).</li><li>Hoffmann, J., Lubbers, D. W., Heise, H. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Applicability+of+the+Kubelka-Munk+theory+for+the+evaluation+of+reflectance+spectra+demonstrated+for+haemoglobin-free+perfused+heart+tissue.">Applicability of the Kubelka-Munk theory for the evaluation of reflectance spectra demonstrated for haemoglobin-free perfused heart tissue.</a> Physics in Medicine and Biology. 43 (12), 3571-3587 (1998).</li><li>Fabel, H., Lubbers, D. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Measurements+of+Reflection+Spectra+of+Beating+Rabbit+Heart+in+Situ.">Measurements of Reflection Spectra of Beating Rabbit Heart in Situ.</a> Biochemische Zeitschrift. 341 (4), 351 (1965).</li><li>Schenkman, K. A., Beard, D. A., Ciesielski, W. A., Feigl, E. O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparison+of+buffer+and+red+blood+cell+perfusion+of+guinea+pig+heart+oxygenation.">Comparison of buffer and red blood cell perfusion of guinea pig heart oxygenation.</a> American Journal of Physiology - Heart and Circulatory Physiology. 285 (5), 1819-1825 (2003).</li><li>Femnou, A. N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intracardiac+light+catheter+for+rapid+scanning+transmural+absorbance+spectroscopy+of+perfused+myocardium:+measurement+of+myoglobin+oxygenation+and+mitochondria+redox+state.">Intracardiac light catheter for rapid scanning transmural absorbance spectroscopy of perfused myocardium: measurement of myoglobin oxygenation and mitochondria redox state.</a> American Journal of Physiology - Heart and Circulatory Physiology. 313 (6), 1199-1208 (2017).</li><li>Kuzmiak-Glancy, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cardiac+performance+is+limited+by+oxygen+delivery+to+the+mitochondria+in+the+crystalloid-perfused+working+heart.">Cardiac performance is limited by oxygen delivery to the mitochondria in the crystalloid-perfused working heart.</a> American Journal of Physiology - Heart and Circulatory Physiology. 314 (4), 704-715 (2018).</li><li>Chess, D. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optical+spectroscopy+in+turbid+media+using+an+integrating+sphere:+mitochondrial+chromophore+analysis+during+metabolic+transitions.">Optical spectroscopy in turbid media using an integrating sphere: mitochondrial chromophore analysis during metabolic transitions.</a> Analytical Biochemistry. 439 (2), 161-172 (2013).</li><li>Lou, Q., Li, W., Efimov, I. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multiparametric+optical+mapping+of+the+Langendorff-perfused+rabbit+heart.">Multiparametric optical mapping of the Langendorff-perfused rabbit heart.</a> Journal of Visualized Experiments. (55), (2011).</li><li>Coburn, R. F., Grubb, B., Aronson, R. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effect+of+cyanide+on+oxygen+tension-dependent+mechanical+tension+in+rabbit+aorta.">Effect of cyanide on oxygen tension-dependent mechanical tension in rabbit aorta.</a> Circulation Research. 44 (3), 368-378 (1979).</li><li>Schenkman, K. A., Marble, D. R., Burns, D. H., Feigl, E. O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Myoglobin+oxygen+dissociation+by+multiwavelength+spectroscopy.">Myoglobin oxygen dissociation by multiwavelength spectroscopy.</a> Journal of Applied Physiology. 82 (1), 86-92 (1997).</li><li>Femnou, A. N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intracardiac+light+catheter+for+rapid+scanning+transmural+absorbance+spectroscopy+of+perfused+myocardium:+measurement+of+myoglobin+oxygenation+and+mitochondria+redox+state.">Intracardiac light catheter for rapid scanning transmural absorbance spectroscopy of perfused myocardium: measurement of myoglobin oxygenation and mitochondria redox state.</a> American Journal of Physiology - Heart and Circulatory Physiology. 313 (6), 1199-1208 (2017).</li><li>Giles, A. V., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Paradoxical+Arteriole+Constriction+Compromises+Cytosolic+and+Mitochondrial+Oxygen+Delivery+in+the+Isolated+Saline-Perfused+Heart.">Paradoxical Arteriole Constriction Compromises Cytosolic and Mitochondrial Oxygen Delivery in the Isolated Saline-Perfused Heart.</a> American Journal of Physiology - Heart and Circulatory. , (2018).</li><li>Matthews, P. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+31P-NMR+study+of+some+metabolic+and+functional+effects+of+the+inotropic+agents+epinephrine+and+ouabain,+and+the+ionophore+R02-+2985+(X537A)+in+the+isolated,+perfused+rat+heart.">A 31P-NMR study of some metabolic and functional effects of the inotropic agents epinephrine and ouabain, and the ionophore R02- 2985 (X537A) in the isolated, perfused rat heart.</a> Biochimica et Biophysica Acta. 720, 163-171 (1982).</li><li>Lewandowski, E. D., Damico, L. A., White, L. T., Yu, X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cardiac+responses+to+induced+lactate+oxidation:+NMR+analysis+of+metabolic+equilibria.">Cardiac responses to induced lactate oxidation: NMR analysis of metabolic equilibria.</a> American Journal of Physiology. 269, 160-168 (1995).</li><li>Lewandowski, E. D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multiplet+structure+of+13C+NMR+signal+from+glutamate+and+direct+detection+of+tricarboxylic+acid+(TCA)+cycle+intermediates.">Multiplet structure of 13C NMR signal from glutamate and direct detection of tricarboxylic acid (TCA) cycle intermediates.</a> Magnetic Resonance in Medicine. 35 (2), 149-154 (1996).</li><li>Ball, D. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hyperpolarized+butyrate:+a+metabolic+probe+of+short+chain+fatty+acid+metabolism+in+the+heart.">Hyperpolarized butyrate: a metabolic probe of short chain fatty acid metabolism in the heart.</a> Magnetic Resonance in Medicine. 71 (5), 1663-1669 (2014).</li><li>Mariotti, E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Modeling+non-linear+kinetics+of+hyperpolarized+[1-(13)C]+pyruvate+in+the+crystalloid-perfused+rat+heart.">Modeling non-linear kinetics of hyperpolarized [1-(13)C] pyruvate in the crystalloid-perfused rat heart.</a> NMR in Biomedicine. 29 (4), 377-386 (2016).</li><li>Pisarenko, O. I., Khlopkov, V. N., Ruuge, E. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+1H+NMR+study+of+succinate+synthesis+from+exogenous+precursors+in+oxygen-deprived+rat+heart+mitochondria.">A 1H NMR study of succinate synthesis from exogenous precursors in oxygen-deprived rat heart mitochondria.</a> Biochemistry International. 12 (1), 145-153 (1986).</li><li>Kuzmiak-Glancy, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cardiac+performance+is+limited+by+oxygen+delivery+to+the+mitochondria+in+the+crystalloid-perfused+working+heart.">Cardiac performance is limited by oxygen delivery to the mitochondria in the crystalloid-perfused working heart.</a> American Journal of Physiology - Heart and Circulatory Physiology. 314 (4), 704-715 (2018).</li><li>Schenkman, K. A., Marble, D. R., Burns, D. H., Feigl, E. O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Myoglobin+oxygen+dissociation+by+multiwavelength+spectroscopy.">Myoglobin oxygen dissociation by multiwavelength spectroscopy.</a> American Journal of Physiology. 82 (1), 86-92 (1997).</li><li>Beard, D. A., Schenkman, K. A., Feigl, E. O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Myocardial+oxygenation+in+isolated+hearts+predicted+by+an+anatomically+realistic+microvascular+transport+model.">Myocardial oxygenation in isolated hearts predicted by an anatomically realistic microvascular transport model.</a> American Journal of Physiology - Heart and Circulatory Physiology. 285 (5), 1826-1836 (2003).</li><li>Paul, R. J., Bohr, D. E., Somlyo, A. P., Sparks, H. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Section+II:+The+Cardiovascular+System,+Vol+II,+Vascular+Smooth+Muscle.">Section II: The Cardiovascular System, Vol II, Vascular Smooth Muscle.</a> Handbook of Physiology. , 201-252 (1980).</li><li>Feigl, E. O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Coronary+physiology.">Coronary physiology.</a> Physiological Reviews. 63, 1-205 (1983).</li></ol>

No conflicts of interest declared.

The isolated retrograde or working perfused heart preparation is a mainstay in the study of cardiac physiology as well as the preclinical investigation of techniques and drugs on the heart. Key to its use has been the ease of preparation, robust functional characteristics and control of experimental parameters as well as the ability to measure many functional parameters of the beating heart. Optical absorbance spectroscopy provides insight into tissue oxygenation as well as mitochondria metabolic activities. Optical spectroscopy has primarily conducted in the isolated perfused heart studies in the reflectance mode that that is difficult to interpret due to motion and light scattering complications.
We have introduced ventricle wall transmission optical spectroscopy (VWTOS) to provide a robust method of monitoring cardiac tissue metabolic chromophores. In a previous publication, we demonstrated that an LED hardwired to the tip of coaxial cable20 makes a unique intracardiac side-firing light source that can be used for VWTOS perfused hearts. The side-firing refers to the projection of light perpendicular to the long axis of the catheter, ideal for illuminating the ventricle free wall. The LED catheter was small enough to not impact cardiac function but required specialized fabrication in the laboratory. The current study presents the use of a 500 micron commercial side-firing catheter that can be coupled to any light source compatible with fiber optics. These side-firing optical catheters were commercially developed for laser ablation perpendicular to the long axis of the fiber. Naturally, we are using light power much lower than required for photoablation. Smaller fibers are available for use on smaller preparations such as the perfused mouse heart27. This fiber optic system provided adequate illumination through the heart wall in the wavelength range where cardiac chromophores absorb (450&#8211;630 nm). Using a pickup fiber optic on the outside of the heart, the absorbance of myoglobin and mitochondria cytochromes can be monitored with excellent temporal and spectral resolution (see Figure 5). The side-firing fiber optic approach has several advantages over the LED catheter for VWTOS, including a much smaller cross-sectional profile of the catheter that minimizes the impact of the catheter on the heart, more flexible reducing impact on cardiac valve and ventricle performance, no electrical connections that can short out in the saline perfusate, and finally a catheter that uses an external light source that increases the flexibility of light source selection for VWTOS.
Due to the strong absorbance of the heart below 490 nm, it is difficult to generate much information on the Soret band of the cytochromes in the region of 410&#8211;445 nm or NADH at 340 nm. Thus, the broad absorbance of FAD at 450 nm is the lowest frequency absorbance that is observed, though the entire absorption peak of this chromophores is not sampled. Using VWTOS the signal to noise ratio is very high as the entire wall is sampled in contrast to surface reflection spectroscopy, commonly used20, which only samples the surface of the heart with numerous scattering issues. VWTOS sampling the entire heart wall is more analogous to Nuclear Magnetic Resonance Spectroscopy (NMRS) measures of many cardiac metabolites such as 31P detected adenosine triphosphate and creatine phosphate28, 13C detected labeled metabolites29,30 including hyperpolarized labels31,32, and 1H detected metabolites33. As the VWTOS can be conducted using non-magnetic devices, it is completely feasible that NMR and VWTOS could be conducted simultaneously. VWTOS is not limited to endogenous chromophores and could be used to monitor absorption from optical probes for pH, Ca2+, and plasma membrane potential.
We use 2 Hz (i.e. 2 samples/sec) which provides excellent single spectrum signal to noise. Though higher sampling rates can be achieved that permit cardiac cycle analysis, previous studies have demonstrated that there&#8217;s no beat to beat variation in the chromophore absorbance, so no effort to selectively collect light as a function of cardiac cycle was made34. Due to the VWTOS geometry, the detection of light is less dependent on tissue motion than reflection methods, since the complex surface scattering events are eliminated. We find that severe motion can disrupt these measures, but the real time spectral analysis quickly reveals spectral transitions inconsistent with tissue chromophore transitions. Again, this only occurs when the heart moves grossly away from the collecting fiber dramatically reducing the amount of collected transmitted light.
The VWTOS data is analyzed using full spectral fitting routine based on a reference library of spectra of cardiac chromophores and the spectrum of the light source as previously described20,22,27,35 with a simple linear least squares approach. This spectral fitting procedure compensated for overlapping absorbance spectrum and does not rely&#160;on &#8220;isobestic&#8221; wavelengths. This full spectrum analysis eliminates artifacts associated with common dual beam (i.e. two wavelength) analysis1,3,6 which has been shown to be problematic20. The added advantage of full spectral analysis is the generation of a goodness of fit from the residuals, not available in dual beam protocols.
In this study, we focused on the effect of cyanide on the optical properties of the heart. As cyanide blocks cytochrome oxidase, it inhibits oxygen consumption and essentially results in a net reduction of all of the cytochromes as the electrons back up in the cytochrome chain. However, the membrane potential apparently remains high, as the redox changes in bL and bH are very small when compared to cytochrome c13. With the cessation of oxygen consumption, the oxygen tension in the tissue should approach the perfusate and we noted an early increase in oxygenated myoglobin with cyanide consistent with the notion that the saline perfused heart, even in retrograde perfusion modes, is not fully oxygenating myoglobin in the cytosol19,20,21,36. Comparing the maximum effect of cyanide on oxygenated myoglobin with the fully deoxygenated spectrum obtained with ischemia reveals a myoglobin oxygenation of only about 88%, consistent with previous studies.
It is important to note in this study that the cyanide effects on myoglobin oxygenation and cytochrome reduction were temporally resolved. It is surprising that the effects of cyanide were first observed on coronary flow and myoglobin before large changes in the heart&#8217;s cytochromes redox state was observed. The early marked increase in flow suggests that an effect on arterial smooth muscle24,37 may occur before gross metabolic effects in the heart cells are observed. The increase in flow, potentially with a modest cyanide induced decrease in respiration, likely results in the immediate increase in oxygenated myoglobin caused by the increase in oxygen delivery. With the spread of the cyanide inhibition to the myocytes, a further increase in coronary flow is observed (see the region marked 3 in Figure 5a), likely driven by numerous metabolic factors38. The large early impact of cyanide on flow suggests that the metabolism of the vascular smooth muscle may be more potent in altering vascular tone than the metabolism of the myocytes. These data support the well-established notion that myoglobin has a much lower affinity for oxygen than COX, even in the intact heart, as myoglobin oxygenation occurred well before changes in mitochondria redox state (Figure 5). This high level of deoxygenated myoglobin under control conditions is consistent with prior studies suggesting that the isolated saline perfused heart may be partially hypoxic even under control conditions9,19,20,21,27,36, underscoring the importance of monitoring cardiac tissue oxygenation when using this important model in cardiac physiology.
We present here the experimental details for conducting transmission absorption spectroscopy on the isolated perfused heart. We have successfully adapted this technique for use on the hearts from the rabbit to the mouse by using a thin side-firing intracardiac optical fiber. Utilizing state of the art full spectral fitting routines, the complex optical interaction of the cardiac chromophores can be easily extracted providing, a near real-time measure of critical elements of myocardial metabolism simultaneously with conventional functional measures.

Optical absorbance spectroscopy for monitoring intact organ biochemistry is a widely-used approach due to its intrinsic, non-destructive nature1,2,3,4,5,6,7,8,9. Myoglobin absorbance provides a measure of the average cytosolic oxygen tension10,11,12. Mitochondrial cytochromes provide information regarding substrate entry at the level of flavins, membrane potential from cytochrome bL:bH13, and oxygen delivery to the mitochondria in the cell from cytochrome oxidase (COX) redox state14. Glancy et al. demonstrated that the activities of each complexes can be determined by measuring the mitochondrial membrane potential and the metabolic rate15. Hence, using optical spectroscopy, a wealth of information can be obtained without the need of exogenous probes or major modifications of current study systems. The goal of this paper is to present a robust method for collecting transmission optical spectra in conventional perfused heart preparations with the only major modification being performing studies in a darkened environment.
Reflectance absorption spectroscopy has been successfully used to perform optical spectroscopy of the perfused heart3,6,16,17,18,19 as well as the heart in vivo1. Reflectance spectroscopy consists of impinging light on the heart surface and collecting the light scattered through the heart as well as diffuse and specular reflected light. Thus, the collected light in this approach is a composite of multiple scattering mechanisms as well as the tissue chromophore absorbances of interest. Due to the motion and complex surface of the heart, the light reflection off the surface of the heart is particularly problematic, altering the depth of penetration and amount of purely reflected light.
The limitations of reflectance absorption spectroscopy presented above were resolved by introducing an optical catheter into the left ventricle cavity, permitting the collection of transmitted light across the left ventricle free wall20. The benefits of transmission spectroscopy for this type of study was appreciated in early invasive studies by Tamura et al.9 The current implementation provides a very robust transmission absorption spectroscopy analysis of the intact heart with regards to cytosolic oxygenation and mitochondria redox state under a variety of conditions21. These initial studies used a specially fabricated catheter with a powered LED on the tip oriented to generate a side-firing pattern of white light through the myocardium. However, the relatively large LED tipped catheter is only appropriate for use on medium size hearts (rabbit, guinea pig, etc.) and required custom fabrication. In the current study, a method of using a commercially available 200-micron core side-firing optical fiber as a light guide is presented. Instead of a wired LED at the tip, the catheter with the 500-micro tip redirects light from an external source increasing the versatility of the system. This approach allows the use of a wide variety of external light sources including lasers for applications such as Raman scattering spectroscopy. To quantify this data, an online full multicomponent spectral analysis using known reference spectra to improve the accuracy of the spectroscopic determination of cardiac chromophores is presented as previously described20,22. The source code for this analysis will be provided by the authors upon request. Using this approach, information on cardiac biochemistry and mitochondrial function can be obtained simultaneously with the conventional cardiac functional parameters with little or no impact on the heart preparation. As the heart is critically dependent on mitochondrial function and oxygen delivery, this technical addition to the classical perfused heart system will greatly improve the interpretation and utility of this important model of cardiac performance.

<table><tbody><tr><td>BIOPAC data acquisition system</td><td>BIOPAC</td><td>MP150</td><td>Analog to digitial conversion</td></tr><tr><td>BIOPAC general purpose transducer amplifiers</td><td>BIOPAC</td><td>DA100C</td><td>Pressure monitoring</td></tr><tr><td>BIOPAC System skin temperature amplifier</td><td>BIOPAC</td><td>SKT100B</td><td>temperature monitoring</td></tr><tr><td>Compact Universal 1- and 2- Channel LED Controllers</td><td>Mightex</td><td>SLC-MA02-U</td><td>External light source power supply</td></tr><tr><td>Disposable pressure sensors</td><td>BIOPAC</td><td>RX104A</td><td>Pressure monitoring</td></tr><tr><td>Dual Syringe, Infusion Pump</td><td>KdScientific</td><td>KDS 200 / 200P LEGACY SYRINGE PUMP</td><td>drug injection</td></tr><tr><td>Flow-through probes</td><td>Transonic</td><td>4PXN</td><td>perusate flow monitoring</td></tr><tr><td>Glass Syringe</td><td>FORTUNA Optima</td><td>30 CC</td><td>Air tight fluid injection</td></tr><tr><td>High power fiber-coupled LED white light source</td><td>Mightex</td><td>Type-A FCS-0000</td><td>External light source</td></tr><tr><td>Perfused heart system</td><td>Radnoti</td><td>120101BEZ</td><td>This system was heavily modified to provide adequate flow (see manuscript)</td></tr><tr><td>Phase fluorimeter</td><td>Ocean Optics</td><td>NeoFox-GT</td><td>oxygen concentration</td></tr><tr><td>Pickup fiber optic</td><td>Thor labs</td><td>BF20HSMA01</td><td>Fiber for collecting transmitted light (pick up fiber)</td></tr><tr><td>PowerLab unit</td><td>AD Instruments</td><td>PowerLab 8/35</td><td>Analog to digitial conversion</td></tr><tr><td>Pressure transducers</td><td>BIOPAC</td><td>TSD104A</td><td>pressure monitoring</td></tr><tr><td>Programming environment</td><td>LABViEW</td><td>N/A</td><td>Software for driving spectrometer, digitiziing data and analysis. Code available on request</td></tr><tr><td>Rapid scanning spectrophotometer</td><td>Ocean Optics</td><td>QE65PRO</td><td>Rapid scanning spectrometer for spectral analysis</td></tr><tr><td>Side firing fiber optic</td><td>Polymicro Technologies Molex, LLC 18019 North 25th Av, Phoenic AZ 85023-1200</td><td>JTFLH200230500/1.5M&amp;nbsp;</td><td>side firing fiber optic 200 microns core&amp;nbsp;</td></tr><tr><td>Sodium cyanide</td><td>Sigma-Aldrich</td><td>380970</td><td>Metabolic inhibitor</td></tr><tr><td>Temperature probe</td><td>BIOPAC</td><td>TSD102A</td><td>temperature monitoring</td></tr><tr><td>Tubing flow modules</td><td>Transonic</td><td>TS410</td><td>perusate flow monitoring</td></tr></tbody></table>

intra-cardiac side-firing light catheter for monitoring cellular metabolism using transmural absorbance spectroscopy of perfused mammalian hearts

Absorbance spectroscopy of cardiac muscle provides non-destructive assessment of cytosolic and mitochondrial oxygenation via myoglobin and cytochrome absorbance respectively. In addition, numerous aspects of the mitochondrial metabolic status such as membrane potential and substrate entry can also be estimated. To perform cardiac wall transmission optical spectroscopy, a commercially available side-firing optical fiber catheter is placed in the left ventricle of the isolated perfused heart as a light source. Light passing through the heart wall is collected with an external optical fiber to perform optical spectroscopy of the heart in near real- time. The transmission approach avoids numerous surface scattering interference occurring in widely used reflection approaches. Changes in transmural absorbance spectra were deconvolved using a library of chromophore reference spectra, providing quantitative measures of all the known cardiac chromophores simultaneously. This spectral deconvolution approach eliminated intrinsic errors that may result from using common dual wavelength methods applied to overlapping absorbance spectra, as well as provided a quantitative evaluation of the goodness of fit. A custom program was designed for data acquisition and analysis, which permitted the investigator to monitor the metabolic state of the preparation during the experiment. These relatively simple additions to the standard heart perfusion system provide a unique insight into the metabolic state of the heart wall in addition to conventional measures of contraction, perfusion, and substrate/oxygen extraction.

The system used is an off the shelf small animal perfusion heart system but was heavily modified for use with a rabbit heart. The modifications were primarily to increase the bore size of all the tubing to assure adequate flow delivery to the rabbit heart. Great care was made to assure, at the perfusion pressures used, the flow rate of the native perfusion system exceeded the flow with heart attached by at least 5-fold. 2&#8211;12 &#181;m pore membrane filters were placed in parallel between the fluid pump and the aortic preload bubble trap chamber to remove any debris from the heart.
Transmitted Light from the Rabbit Heart 
Figure 1 presents the spectrum of the catheter (Figure 1A) and the raw spectrum of the transmitted light from the rabbit heart free wall (Figure 1B). These data reveal a very large attenuation of light in the blue region of the spectrum, however the bands of absorbance from myoglobin and the mitochondrial cytochromes can be directly observed between 490 and 580 nm in the insert. It is important in these studies to assure enough transmitted light is detected in the region from 490 to 630 nm to obtain information on the metabolically responsive cardiac chromophores. The positioning of the external and internal fibers is adjusted prior to saving data to maximize light intensity but not saturate the detector in the 625 nm region.
Reference Reduced minus Oxidized Spectra of Reference Chromophores in the heart. 
Figure 2 presents the reference spectra used to fit the difference spectra collected in these studies. These references include myoglobin, cytochrome aa3 (alternatively cytochrome a605 and cytochrome a607, depending on the type of perturbation22), cytochrome a580, cytochrome bL, cytochrome bH, cytochrome c, cytochrome c1, FAD, an absorbance representation of the incident light (denoted I0, which is used to account for sieved light, that is, photons that went through the tissue without being absorbed), and a line (with varying slope and intercept to account for scattering, not shown in Figure 2). Some spectra are noisy, as the concentration of the pure reference material was very low22.
Time course of reference spectra fits during total experiment 
Figure 3 represents the time course of a typical experiment calculated in step 5.15 of the protocol. This consists of a control phase, followed by a cyanide injection phase, followed by a cyanide washout, followed by a deoxygenation phase, and finally ischemia. The changes in the individual chromophores (myoglobin, cytochrome aa3, and cytochrome c) over time are plotted over time along with coronary flow rate. The optical density change of each chromophore is estimated by multiplying the fit coefficient obtained from the linear least squares routine and the representative peak of the chromophore (or the maximum absorbance of said chromophore). For example, for myoglobin, the fit coefficient of the myoglobin reference is multiplied by the value of the myoglobin reference spectrum at 580 nm. Note the rapid oxygenation of myoglobin to the addition of cyanide is matched by the increase in flow but is before the significant reduction of cytochromes. This effect is partially recovered with the washout of cyanide. Finally, full reduction of cytochromes and deoxygenation of myoglobin is obtained with ischemia. These data demonstrate that dynamic data concerning the metabolic status of the heart can easily be obtained with this methodology. The position of the spectra used for the difference spectra are marked on this time course as: C baseline, CN cyanide injection, CNW cyanide Washout, H N2 Hypoxia (nitrogen being bubbled in the perfusate instead of oxygen), and HI No flow ischemia (no perfusate flowing through the heart).
Difference Spectrum of Cyanide Treatment versus Control and Fit of Cyanide Difference Spectrum from Rabbit Heart. 
To obtain a difference spectrum, two absolute spectra are subtracted. Each absolute spectrum is obtained by taking an average of many (typically 100) spectra to optimize signal to noise ratio. Figure 4A represents the difference spectrum of the control (C) and cyanide (CN) treated heart. Using the reference spectra outlined in Figure 2, the fit spectrum is calculated. The residual spectrum is the subtraction of the fit from the raw data. The same scheme is used for all the subsequent spectral presentations. Figure 4B presents the spectra amplitudes of the reference spectra (shown in Figure 2) used to fit Figure 4A. Strong increases in absorbance of most of the cytochromes are observed as the flow of electrons down the cytochrome chain was blocked by cyanide in the steady state. In addition, the absorbance of oxygenated myoglobin increased as the consumption of oxygen was eliminated by cyanide. Figure 4C presents the difference spectra and fit of the difference spectrum from CNW and CN, revealing the partial reversal of the cyanide effect. This was accomplished by selecting time points in protocol step 5.18, moving T0 to the CNW and T1 to CN region of the time course. Figure 4D presents the difference spectrum of HI and C, which represents the fully deoxygenated and reduced state of the cytosol and mitochondria versus the control condition. Again, this was performed at protocol step 5.18, moving T0 to C and T1 to HI.
Initial time course of cyanide effects on coronary flow and chromophores 
Figure 5A shows an example of the initiation of the cyanide effect on the tissue. The fits for myoglobin, cytochrome a605 and cytochrome c along with the coronary flow are presented for a single heart. These time courses were created at protocol step 5.15 for the cyanide experiment. The individual difference versus the baseline (position 1) are shown in Figure 5B. The spectra were generated from the corresponding position number (1-4) on the time course. This was accomplished at protocol step 5.18, where T0 was always in position 1, and subsequently different spectra (2&#8211;4) were created by moving T1 to position 2, 3, and 4 respectively. Somewhat surprising was the observation that flow and myoglobin oxygenation increased before significant changes in cytochrome redox state. The initiation of the changes in flow and chromophore absorbance were estimated by linear extrapolation of the initial rate of change from the baseline. Using this approach, and setting the change in coronary flow as time zero, the increase in myoglobin oxygenation initiated 1.71 min &#177; 0.39 min after the change in flow, while cytochrome a605 and cytochrome c absorbance were nearly identical but much slower at 4.24 min &#177; 0.76 min and 4.34 min &#177; 0.77 min, respectively (n = 8). These data suggest that cyanide relaxes vascular tone24 before a major change in cardiac muscle metabolic state occurs. This effect is likely caused by cyanide encountering the vascular smooth muscle prior to reaching effective dose around the cardiac myocytes.
Estimates of Myoglobin oxygenation in control hearts 
Using the cyanide data as an estimate of total myoglobin oxygenation and the ischemia data for fully deoxygenated myoglobin, we estimate that under control conditions myoglobin was only 88.2% &#177; 1.0% (n = 10) oxygenated, consistent with prior studies20,21,25.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/58992/58992fig1.jpg" /> 
Figure 1: Spectra of the side-firing optical catheter. (A) This is a spectrum of the emitted light from the remote light source through the catheter detected with the pickup fiber at about 1 cm from the catheter. In this geometry, the heart is absent and the intensity of the light source is tuned so that the detector does not saturate. (B) The side-firing catheter is inserted in the left ventricle and the transmitted light from the heart is collected and shown. The insert shows the 400 to 580 nm region expanded, revealing the complex transmission of light from this region. <a href="https://www.jove.com/files/ftp_upload/58992/58992fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/58992/58992fig2.jpg" /> 
Figure 2: Reference spectra of cardiac chromophores used for spectral fitting. Spectra were collected via a variety of methods22 and are of reduced &#8211; oxidized (for the cytochromes) and deoxygenated &#8211; oxygenated (for myoglobin). For I0, the spectrum in Figure 1A is simply converted to an absorbance term to make the reference. <a href="https://www.jove.com/files/ftp_upload/58992/58992fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/58992/58992fig3.jpg" /> 
Figure 3: Flow and optical changes over time. The optical density change (&#916;OD) of each chromophore is simply the fitted individual chromophore&#8217;s spectrum at its maximum absorbance. The maximum absorbance frequencies were as previously described20,26. The presented time course is for one experiment, showing a baseline, followed by cyanide injection (0.10 mM at maximum perfusate flow), cyanide washout, nitrogen hypoxia performed by replacing oxygen with nitrogen, and then complete ischemia. <a href="https://www.jove.com/files/ftp_upload/58992/58992fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/58992/58992fig4.jpg" /> 
Figure 4: Fitted difference spectra of various conditions. (A) Spectrum of the cyanide injection minus the baseline. The fit spectrum obtained from the least square routine is also plotted. The residual spectrum is the difference between the raw and fit spectra. (B) The reference spectra used to create the fit presented in Figure 4A. The program scales the references in figure 2 to their relative contribution in the current difference spectrum. (C) Same as in A, but showing the difference spectrum of washout versus cyanide injection. (D) Same as in A, but showing the difference spectrum of ischemia versus baseline. <a href="https://www.jove.com/files/ftp_upload/58992/58992fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/58992/58992fig5.jpg" /> 
Figure 5: High temporal resolution of cyanide infusion effect on selected cytochromes, myoglobin and cardiac flow. (A) Time course of cardiac flow, deoxymyoglobin, reduced cytochrome a605, and reduced cytochrome c. Numbers refer to the position of the spectra taken relative to baseline in Figure 5B. (B) Difference spectra for the 4 positions labeled in Figure 5A versus control (position 1). <a href="https://www.jove.com/files/ftp_upload/58992/58992fig5large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Intra-cardiac Side-Firing Light Catheter for Monitoring Cellular Metabolism using Transmural Absorbance Spectroscopy of Perfused Mammalian Hearts at JoVE.com

Intra-cardiac Side-Firing Light Catheter for Monitoring Cellular Metabolism using Transmural Absorbance Spectroscopy of Perfused Mammalian Hearts

Here we introduce a method for using an intra-ventricle optical catheter in perfused hearts to perform absorbance spectroscopy across the heart wall. The data obtained provides robust information on tissue oxygen tension as well as substrate utilization and membrane potential simultaneously with cardiac performance measures in this ubiquitous preparation.

Absorbance spectroscopy of cardiac muscle provides non-destructive assessment of cytosolic and mitochondrial oxygenation via myoglobin and cytochrome ...

intra-cardiac-side-firing-light-catheter-for-monitoring-cellular

Research

JoVE Journal

Bioengineering

6.7K Views.  National Institutes of Health. Dit nieuwe protocol stelt de onderzoeker in staat om meerdere metabole parameters te controleren, waaronder cytosolische en mitochondriale oxygenatie en cytosolische redox-status. Terwijl tegelijkertijd het meten van de traditionele metingen van de myocardfunctie. Het belangrijkste voordeel van deze techniek is een eenvoudige niet-destructieve dynamische monitoring van belangrijke elementen van mitochondriaal metabolisme in het perfused hart dat de artefacten in verband met reflectie spectros...

Video: Intra-cardiac Side-Firing Light Catheter for Monitoring Cellular Metabolism using Transmural Absorbance Spectroscopy of Perfused Mammalian Hearts

Multiparametric Optical Mapping of the Langendorff-perfused Rabbit Heart

Optical imaging and fluorescent probes have significantly advanced research methodology in the field of cardiac electrophysiology in ways that could not have been accomplished by other approaches1. With the use of the calcium- and voltage-sensitive dyes, optical mapping allows measurement of transmembrane action potentials and calcium transients with high spatial resolution without the physical contact with the tissue. This makes measurements of the cardiac electrical activity possible under many conditions where the use of electrodes is inconvenient or impossible1. For example, optical recordings provide accurate morphological changes of membrane potential during and immediately after stimulation and defibrillation, while conventional electrode techniques suffer from stimulus-induced artifacts during and after stimuli due to electrode polarization1. 
The Langendorff-perfused rabbit heart is one of the most studied models of human heart physiology and pathophysiology. Many types of arrhythmias observed clinically could be recapitulated in the rabbit heart model. It was shown that wave patterns in the rabbit heart during ventricular arrhythmias, determined by effective size of the heart and the wavelength of reentry, are very similar to that in the human heart2. It was also shown that critical aspects of excitation-contraction (EC) coupling in rabbit myocardium, such as the relative contribution of sarcoplasmic reticulum (SR), is very similar to human EC coupling3. Here we present the basic procedures of optical mapping experiments in Langendorff-perfused rabbit hearts, including the Langendorff perfusion system setup, the optical mapping systems setup, the isolation and cannulation of the heart, perfusion and dye-staining of the heart, excitation-contraction uncoupling, and collection of optical signals. These methods could be also applied to the heart from species other than rabbit with adjustments to flow rates, optics, solutions, etc.
Two optical mapping systems are described. The panoramic mapping system is used to map the entire epicardium of the rabbit heart4-7. This system provides a global view of the evolution of reentrant circuits during arrhythmogenesis and defibrillation, and has been used to study the mechanisms of arrhythmias and antiarrhythmia therapy8,9. The dual mapping system is used to map the action potential (AP) and calcium transient (CaT) simultaneously from the same field of view10-13. This approach has enhanced our understanding of the important role of calcium in the electrical alternans and the induction of arrhythmia14-16.

This article describes the basic procedures for conducting optical mapping experiments in the Langendorff-perfused rabbit heart using the panoramic imaging system, and the dual (voltage and calcium) imaging modality.

Optical imaging and fluorescent probes have significantly advanced research methodology in the field of cardiac electrophysiology in ways that could not ...

Optical Mapping of Action Potentials and Calcium Transients in the Mouse Heart

The mouse heart is a popular model for cardiovascular studies due to the existence of low cost technology for genetic engineering in this species. Cardiovascular physiological phenotyping of the mouse heart can be easily done using fluorescence imaging employing various probes for transmembrane potential (Vm), calcium transients (CaT), and other parameters. Excitation-contraction coupling is characterized by action potential and intracellular calcium dynamics; therefore, it is critically important to map both Vm and CaT simultaneously from the same location on the heart1-4. Simultaneous optical mapping from Langendorff perfused mouse hearts has the potential to elucidate mechanisms underlying heart failure, arrhythmias, metabolic disease, and other heart diseases. Visualization of activation, conduction velocity, action potential duration, and other parameters at a myriad of sites cannot be achieved from cellular level investigation but is well solved by optical mapping1,5,6. In this paper we present the instrumentation setup and experimental conditions for simultaneous optical mapping of Vm and CaT in mouse hearts with high spatio-temporal resolution using state-of-the-art CMOS imaging technology. Consistent optical recordings obtained with this method illustrate that simultaneous optical mapping of Langendorff perfused mouse hearts is both feasible and reliable.

This paper details the dissection procedure, instrumental setup, and experimental conditions during optical mapping of transmembrane potential (Vm) and intracellular calcium transient (CaT) in intact isolated Langendorff perfused mouse hearts.

The mouse heart is a popular model for cardiovascular studies due to the existence of low cost technology for genetic engineering in this species. ...

High-Resolution Endocardial and Epicardial Optical Mapping in a Sheep Model of Stretch-Induced Atrial Fibrillation

Atrial fibrillation (AF) is a complex cardiac arrhythmia with high morbidity and mortality.1,2 It is the most common sustained cardiac rhythm disturbance seen in clinical practice and its prevalence is expected to increase in the coming years.3 Increased intra-atrial pressure and dilatation have been long recognized to lead to AF,1,4 which highlights the relevance of using animal models and stretch to study AF dynamics. Understanding the mechanisms underlying AF requires visualization of the cardiac electrical waves with high spatial and temporal resolution. While high-temporal resolution can be achieved by conventional electrical mapping traditionally used in human electrophysiological studies, the small number of intra-atrial electrodes that can be used simultaneously limits the spatial resolution and precludes any detailed tracking of the electrical waves during the arrhythmia. The introduction of optical mapping in the early 90's enabled wide-field characterization of fibrillatory activity together with sub-millimeter spatial resolution in animal models5,6 and led to the identification of rapidly spinning electrical wave patterns (rotors) as the sources of the fibrillatory activity that may occur in the ventricles or the atria.7-9 Using combined time- and frequency-domain analyses of optical mapping it is possible to demonstrate discrete sites of high frequency periodic activity during AF, along with frequency gradients between left and right atrium. The region with fastest rotors activates at the highest frequency and drives the overall arrhythmia.10,11 The waves emanating from such rotor interact with either functional or anatomic obstacles in their path, resulting in the phenomenon of fibrillatory conduction.12 Mapping the endocardial surface of the posterior left atrium (PLA) allows the tracking of AF wave dynamics in the region with the highest rotor frequency. Importantly, the PLA is the region where intracavitary catheter-based ablative procedures are most successful terminating AF in patients,13 which underscores the relevance of studying AF dynamics from the interior of the left atrium. Here we describe a sheep model of acute stretch-induced AF, which resembles some of the characteristics of human paroxysmal AF. Epicardial mapping on the left atrium is complemented with endocardial mapping of the PLA using a dual-channel rigid borescope c-mounted to a CCD camera, which represents the most direct approach to visualize the patterns of activation in the most relevant region for AF maintenance.

This report provides a detailed description of the methodology and results of simultaneous endocardial and epicardial optical mapping of electrical excitation in the intact left atrium of a Langendorff-perfused sheep heart during stretch-induced atrial fibrillation.

 Atrial fibrillation (AF) is a complex cardiac arrhythmia with high morbidity and mortality.1,2 It is the most common sustained cardiac rhythm disturbance ...

Medicine

NADH Fluorescence Imaging of Isolated Biventricular Working Rabbit Hearts

Since its inception by Langendorff1, the isolated perfused heart remains a prominent tool for studying cardiac physiology2. However, it is not well-suited for studies of cardiac metabolism, which require the heart to perform work within the context of physiologic preload and afterload pressures. Neely introduced modifications to the Langendorff technique to establish appropriate left ventricular (LV) preload and afterload pressures3. The model is known as the isolated LV working heart model and has been used extensively to study LV performance and metabolism4-6. This model, however, does not provide a properly loaded right ventricle (RV). Demmy et al. first reported a biventricular model as a modification of the LV working heart model7, 8. They found that stroke volume, cardiac output, and pressure development improved in hearts converted from working LV mode to biventricular working mode8. A properly loaded RV also diminishes abnormal pressure gradients across the septum to improve septal function. Biventricular working hearts have been shown to maintain aortic output, pulmonary flow, mean aortic pressure, heart rate, and myocardial ATP levels for up to 3 hours8.
When studying the metabolic effects of myocardial injury, such as ischemia, it is often necessary to identify the location of the affected tissue. This can be done by imaging the fluorescence of NADH (the reduced form of nicotinamide adenine dinucleotide)9-11, a coenzyme found in large quantities in the mitochondria. NADH fluorescence (fNADH) displays a near linearly inverse relationship with local oxygen concentration12 and provides a measure of mitochondrial redox state13. fNADH imaging during hypoxic and ischemic conditions has been used as a dye-free method to identify hypoxic regions14, 15 and to monitor the progression of hypoxic conditions over time10.
The objective of the method is to monitor the mitochondrial redox state of biventricular working hearts during protocols that alter the rate of myocyte metabolism or induce hypoxia or create a combination of the two. Hearts from New Zealand white rabbits were connected to a biventricular working heart system (Hugo Sachs Elektronik) and perfused with modified Krebs-Henseleit solution16 at 37 &deg;C. Aortic, LV, pulmonary artery, and left &amp; right atrial pressures were recorded. Electrical activity was measured using a monophasic action potential electrode. To image fNADH, light from a mercury lamp was filtered (350&plusmn;25 nm) and used to illuminate the epicardium. Emitted light was filtered (460&plusmn;20 nm) and imaged using a CCD camera. Changes in the epicardial fNADH of biventricular working hearts during different pacing rates are presented. The combination of the heart model and fNADH imaging provides a new and valuable experimental tool for studying acute cardiac pathologies within the context of realistic physiological conditions.

The objective is to monitor the mitochondrial redox state of isolated hearts within the context of physiologic preload and afterload pressures. A biventricular working rabbit heart model is presented. High spatiotemporal resolution fluorescence imaging of NADH is used to monitor the mitochondrial redox state of epicardial tissue.

Since its inception by Langendorff1, the isolated perfused heart remains a prominent tool for studying cardiac physiology2. However, it is not well-suited ...

Optical Mapping of Langendorff-perfused Rat Hearts

Optical mapping of the cardiac surface with voltage-sensitive fluorescent dyes has become an important tool to investigate electrical excitation in experimental models that range in scale from cell cultures to whole-organs[1, 2]. Using state-of-the-art optical imaging systems, generation and propagation of action potentials during normal cardiac rhythm or throughout initiation and maintenance of arrhythmias can be visualized almost instantly[1]. The latest commercially-available systems can provide information at exceedingly high spatiotemporal resolutions and were based on custom-built equipment initially developed to overcome the obstacles imposed by more conventional electrophysiological methods[1]. Advancements in high-resolution and high-speed complementary metal-oxide-semiconductor (CMOS) cameras and intensely-bright, light-emitting diodes (LEDs) as well as voltage-sensitive dyes, optics, and filters have begun to make electrical signal acquisition practical for cardiovascular cell biologists who are more accustomed to working with microscopes. Although the newest generation of CMOS cameras can acquire 10,000 frames per second on a 16,384 pixel array, depending on the type of sample preparation, long-established fluorescence acquisition technologies such as photodiode arrays, laser scanning systems, and cooled charged-coupled device (CCD) cameras still have some distinct advantages with respect to dynamic range, signal-to-noise ratio, and quantum efficiency[1, 3]. In the present study, Lewis rat hearts were perfused ex vivo with a crystalloid perfusate (Krebs-Henseleit solution) at 37&deg;C on a modified Langendorff apparatus. After a 20 minute stabilization period, the hearts were intermittently perfused with 11 mMol/L 2,3-butanedione monoxime to eliminate contraction-associated motion during image acquisition. For optical mapping, we loaded hearts with the fast-response potentiometric probe di-8-ANEPPS[4] (5 &mu;Mol/L) and briefly illuminated the preparation with 475&plusmn;15 nm excitation light. During a typical 2 second period of illumination, &gt;605 nm light emitted from the cardiac preparation was imaged with a high-speed CMOS camera connected to a horizontal macroscope. For this demonstration, hearts were paced at 300 beats per minute with a coaxial electrode connected to an isolated electrical stimulation unit. Simultaneous bipolar electrographic recordings were acquired and analyzed along with the voltage signals using readily-available software. In this manner, action potentials on the surface of Langendorff-perfused rat hearts can be visualized and registered with electrographic signals.

This article describes a high temporal and spatial resolution technique to optically image action potential movement on the surface of Langendorff-perfused rat hearts using a potentiometric dye (di-8-ANEPPS).

Optical mapping of the cardiac surface with voltage-sensitive fluorescent dyes has become an important tool to investigate electrical excitation in ...

Biology

Simultaneous Brightfield, Fluorescence, and Optical Coherence Tomographic Imaging of Contracting Cardiac Trabeculae Ex Vivo

In cardiac muscle, intracellular Ca2+ transients activate contractile myofilaments, causing contraction, macroscopic shortening, and geometric&#160;deformation. Our understanding of the internal relationships between these events has been limited because we can neither &#39;see&#39; inside the muscle nor precisely track the spatio-temporal nature of excitation-contraction dynamics. To resolve these problems, we have constructed a device that combines a suite of imaging modalities. Specifically, it integrates a brightfield microscope to measure local changes of sarcomere length and tissue strain, a fluorescence microscope to visualize the Ca2+ transient, and an optical coherence tomograph to capture the tissue&#39;s geometric&#160;changes throughout the time-course of a cardiac cycle. We present here the imaging infrastructure and associated data collection framework. Data are collected from isolated rod-like tissue structures known as trabeculae carneae. In our instrument, a pair of position-controlled platinum hooks hold each end of an ex vivo muscle sample while it is continuously superfused with nutrient-rich saline solution. The hooks are under independent control, permitting real-time control of muscle length and force. Lengthwise translation enables the piecewise scanning of the sample, overcoming limitations associated with the relative size of the microscope imaging window (540 &#181;m by 540 &#181;m) and the length of a typical trabecula (&#62;2000 &#181;m). Platinum electrodes at either end of the muscle chamber stimulate the trabecula at a user-defined rate. We exploit the stimulation signal as a trigger for synchronizing the data from each imaging window to reconstruct the entire sample twitching under steady-state conditions. Applying image-processing techniques to these brightfield imaging data provides tissue displacement and sarcomere length maps. Such a collection of data, when incorporated into an experiment-modeling pipeline, will provide a deeper understanding of muscle contractile homogeneity and heterogeneity in physiology and pathophysiology.

Simultaneous Brightfield, Fluorescence, and Optical Coherence Tomographic Imaging of Contracting Cardiac Trabeculae Ex Vivo

This protocol presents a collection of sarcomere, calcium, and macroscopic geometric&#160;data from an actively contracting cardiac trabecula ex vivo. These simultaneous measurements are made possible by the integration of three imaging modalities.

In cardiac muscle, intracellular Ca2+ transients activate contractile myofilaments, causing contraction, macroscopic shortening, and ...

Optocardiography and Electrophysiology Studies of Ex Vivo Langendorff-perfused Hearts

Small animal models are most commonly used in cardiovascular research due to the availability of genetically modified species and lower cost compared to larger animals. Yet, larger mammals are better suited for translational research questions related to normal cardiac physiology, pathophysiology, and preclinical testing of therapeutic agents. To overcome the technical barriers associated with employing a larger animal model in cardiac research, we describe an approach to measure physiological parameters in an isolated, Langendorff-perfused piglet heart. This approach combines two powerful experimental tools to evaluate the state of the heart: electrophysiology (EP) study and simultaneous optical mapping of transmembrane voltage and intracellular calcium using parameter sensitive dyes (RH237, Rhod2-AM). The described methodologies are well suited for translational studies investigating the cardiac conduction system, alterations in action potential morphology, calcium handling, excitation-contraction coupling and the incidence of cardiac alternans or arrhythmias.

The objective of this study was to establish a method for investigating cardiac dynamics using a translational animal model. The described experimental approach incorporates dual-emission optocardiography in conjunction with an electrophysiological study to assess electrical activity in an isolated, intact porcine heart model.

Small animal models are most commonly used in cardiovascular research due to the availability of genetically modified species and lower cost compared to ...

Advanced Cardiac Rhythm Management by Applying Optogenetic Multi-Site Photostimulation in Murine Hearts

Ventricular tachyarrhythmias are a major cause of mortality and morbidity worldwide. Electrical defibrillation using high-energy electric shocks is currently the only treatment for life-threatening ventricular fibrillation. However, defibrillation may have side-effects, including intolerable pain, tissue damage, and worsening of prognosis, indicating a significant medical need for the development of more gentle cardiac rhythm management strategies. Besides energy-reducing electrical approaches, cardiac optogenetics was introduced as a powerful tool to influence cardiac activity using light-sensitive membrane ion channels and light pulses. In the present study, a robust and valid method for successful photostimulation of Langendorff perfused intact murine hearts will be described based on multi-site pacing applying a 3 x 3 array of micro light-emitting diodes (micro-LED). Simultaneous optical mapping of epicardial membrane voltage waves allows the investigation of the effects of region-specific stimulation and evaluates the newly induced cardiac activity directly on-site. The obtained results show that the efficacy of defibrillation is strongly dependent on the parameters chosen for photostimulation during a cardiac arrhythmia. It will be demonstrated that the illuminated area of the heart plays a crucial role for termination success as well as how the targeted control of cardiac activity during illumination for modifying arrhythmia patterns can be achieved. In summary, this technique provides a possibility to optimize the on-site mechanism manipulation on the way to real-time feedback control of cardiac rhythm and, regarding the region specificity, new approaches in reducing the potential harm to the cardiac system compared to the usage of non-specific electrical shock applications.

This work reports a method for controlling the cardiac rhythm of intact murine hearts of transgenic channelrhodopsin-2 (ChR2) mice using local photostimulation with a micro-LED array and simultaneous optical mapping of epicardial membrane potential.

Ventricular tachyarrhythmias are a major cause of mortality and morbidity worldwide. Electrical defibrillation using high-energy electric shocks is ...

Optical Mapping of Intra-Sarcoplasmic Reticulum Ca2+ and Transmembrane Potential in the Langendorff-perfused Rabbit Heart

Sarcoplasmic reticulum (SR) Ca2+ handling plays a key role in normal excitation-contraction coupling and aberrant SR Ca2+ handling is known to play a significant role in certain types of arrhythmia. Because arrhythmias are spatially distinct, emergent phenomena, they must be investigated at the tissue level. However, methods for directly probing SR Ca2+ in the intact heart remain limited. This article describes the protocol for dual optical mapping of transmembrane potential (Vm) and free intra-SR [Ca2+] ([Ca2+]SR) in the Langendorff-perfused rabbit heart. This approach takes advantage of the low-affinity Ca2+ indicator Fluo-5N, which has minimal fluorescence in the cytosol where intracellular [Ca2+] ([Ca2+]i) is relatively low but exhibits significant fluorescence in the SR lumen where [Ca2+]SR is in the millimolar range. In addition to revealing SR Ca2+ characteristics spatially across the epicardial surface of the heart, this approach has the distinct advantage of simultaneous monitoring of Vm, allowing for investigations into the bidirectional relationship between Vm and SR Ca2+ and the role of SR Ca2+ in arrhythmogenic phenomena.

Optical Mapping of Intra-Sarcoplasmic Reticulum Ca2+ and Transmembrane Potential in the Langendorff-perfused Rabbit Heart

This article describes the detailed protocol and equipment necessary for dual optical mapping of transmembrane potential (Vm) and free intra-sarcoplasmic reticulum (SR) Ca2+ in the Langendorff-perfused rabbit heart. This method allows for direct observation and quantification of Vm and SR Ca2+ dynamics in the intact heart.

Sarcoplasmic reticulum (SR) Ca2+ handling plays a key role in normal excitation-contraction coupling and aberrant SR Ca2+ handling is known to play a ...

Local Field Fluorescence Microscopy: Imaging Cellular Signals in Intact Hearts

In the heart, molecular signaling studies are usually performed in isolated myocytes. However, many pathological situations such as ischemia and arrhythmias can only be fully understood at the whole organ level. Here, we present the spectroscopic technique of local field fluorescence microscopy (LFFM) that allows the measurement of cellular signals in the intact heart. The technique is based on a combination of a Langendorff perfused heart and optical fibers to record fluorescent signals. LFFM has various applications in the field of cardiovascular physiology to study the heart under normal and pathological conditions. Multiple cardiac variables can be monitored using different fluorescent indicators. These include cytosolic [Ca2+], intra-sarcoplasmic reticulum [Ca2+] and membrane potentials. The exogenous fluorescent probes are excited and the emitted fluorescence detected with three different arrangements of LFFM epifluorescence techniques presented in this paper. The central differences among these techniques are the type of light source used for excitation and on the way the excitation light is modulated. The pulsed LFFM (PLFFM) uses laser light pulses while continuous wave LFFM (CLFFM) uses continuous laser light for excitation. Finally, light-emitting diodes (LEDs) were used as a third light source. This non-coherent arrangement is called pulsed LED fluorescence microscopy (PLEDFM).

In the heart, molecular events coordinate the electrical and contractile function of the organ. A set of local field fluorescence microscopy techniques presented here enables the recording of cellular variables in intact hearts. Identifying mechanisms defining the cardiac function is critical in understanding how the heart works under pathological situations.

Intra-cardiac Side-Firing Light Catheter for Monitoring Cellular Metabolism using Transmural Absorbance Spectroscopy of Perfused Mammalian Hearts

Samenvatting

Meer video's verkennen